Solubility, flow column method

The flow column method has useful advantages. Manipulation of the system prior to analysis is minimized, and so problems such as adsorption or evaporation that may arise from separation of the saturated solution and the undissolved solute are reduced. The method is rapid and precise [22,33,34], and it is valuable for sparingly soluble systems, such as hydrophobic solutes in water. 

Two general methods, the analytical method and the synthetic method (Grant and Brittain 1995), are available for determining solubility. In the analytical method, the temperature of equilibration is hxed, while the concentration of the solute in a saturated solution is determined at equilibrium by a suitable analytical procedure. The analytical method can be either the traditional, common batch agitation method, or the more recent flow column method. In the synthetic method, the composition of the solute-solvent system is hxed by appropriate addition and mixing of the solute and solvent, then the temperature at which the solid solute just dissolves or just crystallizes is carefully bracketed. 

Obviously, cyanide cannot be directly measured by flame atomic absorption spectrometry (FAAS), but an indirect approach, as that schematically depicted in Figure 7.16, allows this possibility to be implemented, improving detection limits with regard to those reported previously for flow-based methods. The FIA manifold relies on the formation of soluble metal-cyanide complexes as the sample passes through a small column packed with soUd-phase reagent (SPR). Different SPR have been tested for indirect determination of cyanide using FIA. In all cases the eluted complex is measured by FAAS. Detection limits close to 0.05 mg/1 cyanide have been reported [28]. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

In a solubility experiment the solubility of the compound of interest is measured in the presence and absence of dissolved humic materials. Two techniques were used to measure solubility a shake and filter method similar to that used by Yalkowsky, and a flow through column technique similar to that used by May et al. 9 The measured solubilities of a number of compounds in our experiments were always higher in the presence of humic materials. This increase in the solubility is due to the binding of the compound by humic materials. In the presence of humic materials the measured solubility consists of two fractions free and bound. The free concentration should be the same in the presence or absence of humic materials. The difference between the solubilities of the compound in the presence and absence of humic materials is therefore a measurement of the bound fraction. 

Reservoir method This method makes use of the displacement principle. Brine or any other saturated solution in which a gas has low solubility is used as the liquid. Gas from the column is collected in a burette from which the displaced liquid flows to a reservoir. As the gas collection proceeds, the gas is collected under increasing pressure conditions, thereby changing the flow rate as well as the frequency of bubble formation. In order to collect gas under atmospheric conditions, the levels of the liquid in the burette and the reservoir must always be kept equal. This requires manual adjustments. 

Column packing materials such as silica gel contain a large amount of water, and separation involves partition between an immobilized aqueous phase in the gel and a mobile, often organic, solvent flowing through the column. Usually materials elute sooner when they are more soluble in the mobile phase than in the aqueous phase. These methods are closely related to perfusion chromatography, which is described in Section 2. 

Ross and Riley (11) reported an HPLC method for determination of solubilities of lomefloxacin and other fluoroquinolones. An MOS Hypersil (C8) reversed-phase column (5 pm, 15 cm x 4.6 mm) was used with UV detection at 280 nm. The mobile phase was tetrahydrofuran-acetonitrile-H3PC>4 (10 mM)-triethylamine (10 30 60 0.03 by volume) with a flow rate of 1.5 mL/min. 

The column generator (and related HPLC) method of determining aqueous solubility has become the predominant method in the literature over the past 5 years. In this method, a column is packed with an inert solid support which provides a high surface area for the solute to insure quick equilibration between it and the aqueous phase. After the support is impregnated with the solute of interest and excess solute has been displaced, the column is brought to the desired temperature and the aqueous concentration is determined as a function of flow rate. The equilibrium concentration corresponds to the highest eluting flow rate where solute concentration remains flow-independent. 

---